Department of Biomedical Data Science, Molecular and Systems Biology and the Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth College, Hanover, NH, United States of America.

Abstract

Wnt/β-catenin signal transduction directs intestinal stem cell (ISC) proliferation during homeostasis. Hyperactivation of Wnt signaling initiates colorectal cancer, which most frequently results from truncation of the tumor suppressor Adenomatous polyposis coli (APC). The β-catenin-TCF transcription complex activates both the physiological expression of Wnt target genes in the normal intestinal epithelium and their aberrantly increased expression in colorectal tumors. Whether mechanistic differences in the Wnt transcription machinery drive these distinct levels of target gene activation in physiological versus pathological states remains uncertain, but is relevant for the design of new therapeutic strategies. Here, using a Drosophila model, we demonstrate that two evolutionarily conserved transcription cofactors, Earthbound (Ebd) and Erect wing (Ewg), are essential for all major consequences of Apc1 inactivation in the intestine: the hyperactivation of Wnt target gene expression, excess number of ISCs, and hyperplasia of the epithelium. In contrast, only Ebd, but not Ewg, mediates the Wnt-dependent regulation of ISC proliferation during homeostasis. Therefore, in the adult intestine, Ebd acts independently of Ewg in physiological Wnt signaling, but cooperates with Ewg to induce the hyperactivation of Wnt target gene expression following Apc1 loss. These findings have relevance for human tumorigenesis, as Jerky (JRK/JH8), the human Ebd homolog, promotes Wnt pathway hyperactivation and is overexpressed in colorectal, breast, and ovarian cancers. Together, our findings reveal distinct requirements for Ebd and Ewg in physiological Wnt pathway activation versus oncogenic Wnt pathway hyperactivation following Apc1 loss. Such differentially utilized transcription cofactors may offer new opportunities for the selective targeting of Wnt-driven cancers.

Ewg is not required for intestinal homeostasis in physiological conditions.

(A-B’) No overt change in the number of progenitor cells (marked with esg>GFP, green A and B) is detected in ewgP1 mutants compared to controls, suggesting that Ewg is not required for homeostasis of intestinal tissues under physiological conditions. In addition, ewgP1 mutants exhibit a normal, well-organized epithelial structure (marked with Arm, magenta A’ and B’). (C-D) Similarly, no obvious change in the number of progenitor cells is detected in ewg2/ewg1 mutants compared to controls. Progenitor cells are identified as small cells with strong Arm staining and the absence of Prospero staining. (E) Quantification of progenitor cell numbers in a defined field in the posterior midguts of age matched ewg2/+ and ewg2/ewg1 flies. ns: not significant (t-test). (F) Cell type specific RNA-seq of Drosophila intestines (performed by Dr. Buchon’s lab; FlyGut-seq []) revealed that under homeostatic conditions, ebd1 is expressed in all gut cell types, whereas ewg is expressed at very low levels in the gut. RPKM: Reads Per Kilobase of transcript per Million. (G) Quantification of Ewg expression levels in control and Apc1 mutant guts. Scale bars: (A-D) 10 μm. Genotypes: (A-B’) control:esg-Gal4 UAS-GFP/+ewgP1: ewgP1; esg-Gal4 UAS-GFP/+ (G) control:Apc1Q8/+Apc1 mutant:Apc1Q8.

(A) Ebd, but not Ewg, is required for Wingless-dependent regulation of midgut homeostasis under physiological conditions. (B) Both Ebd and Ewg are required for high-level transcription of Wingless target genes and consequent ISC overproliferation and intestinal hyperplasia following Apc1 inactivation.